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Article

Effects of Anthracnose on the Structure and Diversity of Endophytic Microbial Communities in Postharvest Avocado Fruits

by
Xi Chen
1,
Zhuoen Jiang
1,
Peng He
1,
Xiuhua Tang
1,
Haiyun Song
1,
Tao Zhang
1,
Zhejun Wei
1,
Tao Dong
2,
Shufang Zheng
1,
Xinghao Tu
3,
Jian Qin
2,
Jingjing Chen
3,* and
Wenlin Wang
1,*
1
Guangxi South Subtropical Agricultural Sciences Research Institute, Guangxi Academy of Agricultural Sciences, Longzhou 532415, China
2
Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
3
Key Laboratory of Tropical Fruit Biology, Ministry of Agriculture & Rural Affairs, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524091, China
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(11), 2487; https://doi.org/10.3390/agronomy14112487
Submission received: 26 August 2024 / Revised: 20 October 2024 / Accepted: 21 October 2024 / Published: 24 October 2024
(This article belongs to the Section Horticultural and Floricultural Crops)
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Versions Notes

Abstract

:
This study aimed to provide foundational research for the biological control of postharvest avocado fruits anthracnose and establish a microbial system of postharvest avocado fruits. The high-throughput sequencing of avocado fruits from the anthracnose-infected and healthy groups was performed using Illumina NovaSeq second-generation sequencing technology. The results revealed that, except for Colletotrichum sp. strain 38#, there were differences in the bacterial community structure of avocados before and after infection, as determined through alpha and beta diversity analysis. Additionally, there were significant differences in the endophytic fungal community structure, allowing clear differentiation between the infected and healthy avocados. The endophytic bacterial community was primarily composed of 4 phyla and 10 genera, with the Bacteroidota phylum and Chryseobacterium genus demonstrating sensitivity to anthracnose pathogens, as evidenced by a decrease in their relative abundance after infection. The endophytic fungal community was characterized by 3 phyla and 10 genera. After infection, the relative abundance of 2 phyla (Anthophyta and Basidiomycota) and 7 genera (Eucalyptus, Candida, Kluyveromyces, Talaromyces, Oidiodendron, Nigrospora, and Pestalotiopsis) decreased, whereas the relative abundance of the Colletotrichum genus increased dramatically. The LEfSe (Linear discriminant analysis Effect Size) analysis indicated that significant biomarkers were more prevalent in endophytic bacteria than in endophytic fungi in the avocados. In endophytic bacteria, the key biomarkers included the Firmicutes phylum (Bacilli class), Proteobacteria phylum (Gammaproteobacteria class, Pseudomonadales order, Pseudomonadaceae family, and Pseudomonas genus), Flavobacteriales order, Weeksellaceae family, and Chryseobacterium genus. In endophytic fungi, the important biomarkers were Saccharomycetes class (Saccharomycetales order), Glomerellales order (Glomerellaceae family and Colletotrichum genus), and Botryosphaeriales order (Botryosphaeriaceae family and Lasiodiplodia genus). These results may provide a theoretical basis for the development of future biological agents for avocado anthracnose.

1. Introduction

Avocado (Persea americana Mill) is a fruit rich in fats, fatty acids, dietary fiber, protein, and minerals, and it holds high economic values in international trade [1,2]. It is cultivated in the Hainan, Guangdong, Guangxi, Taiwan, and Yunnan provinces in China and has rapidly developed as a valuable tropical fruit tree in recent years. However, avocados face postharvest storage losses primarily due to the anthracnose diseases caused by various fungi, including Colletotrichum aenigma, C. alienum, C. fructicola, C. gloeosporioides sensu stricto, C. karstii, C. nupharicola, C. siamense, C. theobromicola, C. perseae, and others [3]. Currently, the control of avocado anthracnose relies mainly on the synthetic fungicides, but previous research has reported the inability to control fungal diseases due to the occurrence of fungicide-tolerant strains of pathogens [4,5]. Also, pesticide residue contamination compromises avocado safety [6,7]. Therefore, there is an urgent need to develop the new environmentally friendly biological control methods [8,9]. The first step towards a more sustainable disease management system involves collecting comprehensive information on all aspects of the ecosystem [10]. However, there has been limited research on the interactions between postharvest plant hosts, pathogens, environmental factors, and postharvest plant microbiota [11]. Gaining insight into microbial population dynamics in plant systems offers new opportunities for a more comprehensive approach to disease control [12]. Endophytic bacteria and endophytic fungi with antagonistic effects present a promising option for controlling postharvest avocado anthracnose, as they have shown potential in similar applications to some plants [13,14].
Endophytes are defined as organisms that inhabit plant organs and can colonize internal plant tissues at some point in their life without causing apparent harm to their host [15]. Plants are rich in endophytic flora that can promote growth and enhance resilience [16,17,18]. Endophytes also control plant diseases by competing with pathogenic bacteria for ecological niches and secreting antimicrobial substances [19,20]. Moreover, the fruit surface is colonized by complex and often resilient microbial communities [21]. Leveraging the endophytic flora of plants can help control diseases and avoid pesticide residues. However, there have been few studies on postharvest endophytes in avocados, both domestically and internationally. Some studies have focused on the fungal development level in freshly harvested avocado fruits and the changes in endophytic flora in the roots and branches of avocado trees [10,22,23,24]. However, the literature on the differential structure of endophytic flora before and after anthracnose infection in avocados is lacking, although similar studies have been conducted on cherry fruits, navel orange fruits, fresh-cut dragon fruits, and mulberry fruits [25,26,27]. These studies have revealed differences in endophytic structures between susceptible and healthy plants. The diversity of endophytic fungi was significantly lower in susceptible fruits, indicating that the structure and diversity of endophytic plant colonies are related to pathogen invasion [28]. Identifying the changes in the structural diversity of endophytes in avocado fruits before and after anthracnose infection may lead to new breakthroughs in the biological control of avocado diseases.
In this study, high-throughput sequencing of postharvest avocado fruits from anthracnose-infected and healthy groups was conducted using Illumina NovaSeq second-generation sequencing technology. This study focused on the changes in the structure and diversity of fruit endophyte colonies before and after infection. The interactions between endophytes and anthracnose pathogens were analyzed, and the findings could provide a theoretical basis for the biological control of anthracnose.

2. Materials and Methods

2.1. Materials and Treatments

The avocado fruits (cv. ‘Hass’) that reached the physiological maturity stage (dry matter content, 25.6%) were adopted as the experimental materials. Fruits with consistent size and without defects were selected. They were randomly divided into healthy (CK) and infected groups. Three infected groups comprised the avocados inoculated with three Colletotrichum sp. strains (31#, 38#, and 64#). The method of prick inoculation was adopted [29]. Two stab wounds were evenly stabbed on each fruit surface. The solid medium with Colletotrichum sp. strain (Φ = 5 mm) was inoculated on the stab wounds and placed in the fresh-keeping box together with wet absorbent cotton to achieve the purpose of moisturizing. All the samples (4 fruits in each group, 4 groups, 16 fruits in total) were incubated at 28 °C for 7 d, after which the avocado rind and the pulp from both the healthy and infected groups were collected and stored in a −80 °C refrigerator for further experiments. The details of inoculation are shown in Figure 1 and Figure 2. The Colletotrichum sp. strains used were extracted from avocados with anthracnose disease, which were previously identified in our laboratory.

2.2. Methods

2.2.1. DNA Extraction and Polymerase Chain Reaction (PCR) Amplification

The above groups of samples were fully mixed and ground by liquid nitrogen, and 80 mg of powder was weighed for use. Genomic DNA was extracted from the samples using a BioFastSpin Plant Genomic DNA Extraction Kit (BSC18S1, Bioer Technology, Hangzhou, China) according to the manufacturer’s instructions. The purity and concentration of DNA were assessed using agarose gel electrophoresis (Power source: Dyy-BC, Electrophoresis bath: DYCP-31DN, Beijing Liuyi Biotechnology Co., Ltd., Beijing, China). Briefly, the V5-V7 16S rRNA region was amplified using primers 799F (AACMGGATTAGATACCCKG) and 1193R (ACGTCATCCCCACCTTCC). The ITS2 region was amplified by using a conventional primer pair of ITS3-2024F (GCATCGATGAAGAACGCAGC) and ITS4-2409R (TCCTCCGCTTATTGATATGC). PCR was performed using Ultra HiFidelity PCR Kit (Tiangen, Beijing, China) on a Bio-Rad T100 thermal cycler (Bio-Rad, Hercules, CA, USA) to ensure amplification efficiency and accuracy. PCR products were analyzed using agarose gel electrophoresis (2% mass fraction) and an Ultramicro spectrophotometer (Nanodrop2000, Thermo Fisher, Waltham, MA, USA). The aliquots of PCR products were thoroughly mixed. Then, the Universal DNA Purification Kit (Tiangen, Beijing, China) was used for purification according to the manufacturer’s instructions.

2.2.2. Library Construction and Sequencing

The library construction was performed using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The libraries were quantified using Qubit (Qubit 3.0 Fluorometer, Thermo Fisher, Waltham, MA, USA). Once the libraries were confirmed to be of adequate quality, they were subjected to online sequencing using an Illumina NovaSeq 6000 sequencing machine (Illumina, San Diego, CA, USA).

2.2.3. Processing of Sequencing Data

Based on the barcode sequences and PCR amplification primer sequences, the data for each sample were separated from the downstream data. The sample reads were spliced using FLASH software (V1.2.11, Johns Hopkins University, Baltimore, MD, USA) [30] after truncating the barcode and primer sequences. The Raw Tags were then subjected to the quality control using fastp software (V0.23.4, HaploX Biotechnology, Shenzhen, China) [31], resulting in the high-quality Clean Tags. Finally, the Clean Tags were compared to the database using vsearch software (V2.22.1, Department of Informatics, University of Oslo, Oslo, Norway) [32], and the chimeras were detected and removed, yielding the effective tags.

2.2.4. ASVs Noise Reduction and SPECIES Annotation

The following experiments were carried out according to Bolyen’s method [33] and Schloss’s method [34]. In short, the DADA2 module (v1.26.0) of QIIME 2 software (v2023.2, Northern Arizona University, Flagstaf, AZ, USA) was used for denoising, and sequences with an abundance of less than 5 were be filtered out to obtain the final ASVs (Amplicon Sequence Variants). The ASVs obtained by using Mothur software (v1.48, Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA) were compared with the SSUrRNA database (SILVA138.1) and the UNITE database (v9.0) to obtain species information for each ASV. The taxonomic information was obtained, and the community composition of each sample was calculated at the levels of phylum, class, order, family, genus, and species. MAFFT software (v7.520, Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan) [35] was used for sequence alignment, and the systematic relationships of all ASVs represent sequences were received. Finally, the data of each sample were homogenized and analyzed as follows.

2.2.5. Analysis of Alpha Diversity and Beta Diversity

This method was adopted for sample complexity analysis. The indices of chao1, Shannon, Simpson, ACE, and coverage were calculated using phyloseq (v1.40.0) and vegan (v2.6-8) of R software (v4.4.0, University of Auckland, Auckland, New Zealand), and rarefaction curve and species accumulation boxplot were plotted. NMDS was analyzed using the phyloseq software package of R software (v4.4.0). LEfSe analysis was conducted using LEfSe software (v1.1.2, Harvard University, Cambridge, MA, USA), and the screening value for LDA Score was set to 4.

3. Results

3.1. Analysis of Sequencing Data

The data obtained from the Illumina NovaSeq sequencing platform underwent splicing, quality control, and chimeric filtering, resulting in the Effective Tags for subsequent analysis. On average, there were 102,678; 118,839; 109,913; and 133,205 Effective Tags of bacteria in the CK, 31#, 38#, and 64# groups, respectively. For fungi, there were 127,807; 96,620; 131,409; and 128,802 Effective Tags in the same groups. The specific data are detailed in Table 1 and Table 2. Figure 3 illustrates that the length of endophytic bacterial sequences was predominantly within the 360 to 380 bp range, whereas the endophytic fungal sequences were mainly within the 300 to 320 bp range. The Effective Tags were clustered at the 100% similarity level. In Figure 4, on average, there were 474, 825, 764, and 745 ASVs for bacteria and 304, 209, 161, and 216 ASVs for fungi in the CK, 31#, 38#, and 64# groups, respectively. And the numbers of ASVs regarding kingdom, phylum, class, order, family, genus, species for bacteria and fungi in every group are shown in Figure 4, too. Significant differences between the CK group (healthy avocados) and the infected groups were observed (p < 0.05). The rarefaction curve, depicted in Figure 5, indicated the adequacy of the sequencing data and species richness, with a flattening curve suggesting reasonable data amounts. The species accumulation boxplot, shown in Figure 6, assessed whether the sample size was sufficient, with a gradually rising plot indicating adequate sampling and readiness for data analysis.

3.2. Diversity Analysis of Endophytes

3.2.1. Analysis of Alpha Diversity

Alpha diversity was applied to analyze the microbial community diversity within the sample, reflecting both the richness and diversity. In Table 3 and Table 4, the chao1 index estimated the total number of species present in a community sample, with higher values indicating more low-abundance species. The ACE index also indicated the species richness, with higher values indicating greater community richness. The Shannon index assessed the species richness and evenness, with higher values representing greater community diversity. The Simpson index characterized the species distribution diversity and evenness, with a higher index indicating better species evenness.
The coverage rates of endophytic sequences exceeded 99.90%, indicating that they accurately represented the true endophytic sequences in avocados. For bacteria, all details are shown in Table 3; the chao1 and ACE indices in the CK group were significantly lower than those in the infected groups, whereas the opposite trend was observed for fungi. The total number and richness of endophytic bacteria were higher than those of endophytic fungi. In Table 4, the Shannon and Simpson indices were significantly higher in the CK groups for fungi than in the infected groups, but no such differences were observed for bacteria. Additionally, the diversity and uniformity of bacteria in the infected groups were higher than those of fungi. This suggests that the diversity and richness of endophytic bacteria in healthy avocados were lower than those in diseased avocados, whereas the endophytic fungi did not exhibit such a pattern. Overall, the diversity of endophytic bacteria in both healthy and diseased avocados was greater than that of endophytic fungi.

3.2.2. Analysis of Beta Diversity

Beta diversity was employed to analyze the microbial community composition of the samples and assess their differences. Non-metric multidimensional scaling (NMDS), a ranking method suitable for ecological research, was employed to address the limitations of linear models such as principal component analysis and principal coordinates analysis, thereby better reflecting the nonlinear structure of ecological data [36], as shown in Figure 7. For the endophytic bacteria (Figure 7a), although each sample exhibited independence, there was a notable difference between the groups infected with pathogenic bacteria 31# and 64# and CK groups, whereas 38# did not present significant differences. This indicates that different pathogens can alter the original colony structure of healthy fruits to varying degrees. For the endophytic fungi (Figure 7b), a clear separation was observed between the CK group and the infected group despite the independence of samples within each group, highlighting the significant differences.

3.3. Composition of Endophyte Community Structure

In this study, four groups of samples were analyzed for their community structure at the phylum and genus levels (Figure 8). For the endophytic bacteria, four major phyla were identified in Figure 8a. Proteobacteria was the dominant phylum across all the samples, with Bacteroidota being relatively dominant in the CK groups. In the infected groups, the relatively dominant phyla varied: Firmicutes in Colletotrichum sp. strain 31# and Bacteroidota in the strains 38# and 64#. Eleven major genera are identified in Figure 8b, revealing the distinct microbial community structures among the groups. In the CK group, Stenotrophomonas and Pseudomonas were the dominant genera, with Chryseobacterium being relatively dominant. In group 31#, Stenotrophomonas and Pseudomonas were dominant, while Paenibacillus was relatively dominant. In group 38#, Pseudomonas was the dominant genus, with Stenotrophomonas was relatively dominant. And in 64# group, Stenotrophomonas was the dominant genus, while Ochrobactrum genera were relatively dominant. These findings indicate that although these were both anthracnose pathogens, the avocado fruits infected by different pathogens exhibited distinct colony structures. In contrast, the diversity of endophytic bacteria in the CK groups was significantly lower than that in the infected groups, as detailed in Table 5 and Table 6. These results are consistent with those observed in Section 3.2.1 and Section 3.2.2. The pathogens significantly affected the phyla Bacteroidota, Firmicutes, and Actinobacteriota, with Bacteroidota showing a decreasing trend, whereas Firmicutes and Actinobacteriota showed an increasing one. At the genus level, the structure varied considerably across all groups. Owing to the influence of pathogenic fungi, the abundance of Chryseobacterium decreased, while that of Bacillus increased, with other genera showing inconsistent changes. This variability may be related to the extent of pathogen invasion or individual differences in sample susceptibility.
Among the endophytic fungi, 3 major phyla and 10 major genera were identified, as shown in Table 7 and Table 8 and Figure 9a,b. Ascomycota was the dominant phylum in all samples, whereas Anthophyta was relatively dominant in the CK groups but not in the infected groups. At the genus level, Eucalyptus and Candida were dominant and relatively dominant, respectively, in the CK groups. In the infected groups, Colletotrichum and Lasiodiplodia were the dominant and relatively dominant genera, respectively. The structure of endophytic fungal colonies was similar within the infected groups, and their diversity was significantly lower than that in the CK groups, consistent with the results of Section 3.2.1 and Section 3.2.2. At the phylum level, the abundance of Ascomycota increased, while the abundance of Anthophyta and Basidiomycota decreased significantly in the infected groups compared to that in the CK group. At the genus level, the variation was pronounced, with the exception of the increased abundance of Colletotrichum and Lasiodiplodia, as other genera largely disappeared from the infected groups.

3.4. Analysis of Differences Between Groups of Endophytes

The LEFSe method was employed to analyze differences in endophytes before and after the avocado infection, allowing for the identification of the biomarkers that were statistically different between groups [37]. In the LEFSe cladogram of endophytes, circles radiating from the center outward represent taxonomic levels from phylum to genus. Each small circle at a different taxonomic level denotes a taxon, with the circle diameter proportional to its relative abundance. Species without the significant differences are uniformly colored in yellow. The differential species biomarkers are highlighted by coloring, with the red nodes indicating the microbial taxa that are significant in the red grouping and the green nodes indicating those significant in the green grouping; the details are shown in Figure 10a,b.
Figure 10a and Figure 11a illustrate the significant endophytic bacterial taxa in each group. The biomarkers in the CK groups were substantially lower than those in the infected groups. Specifically, in the CK groups, one phylum (Bacteroidota) with its branches (one class, Bacteroidia; one order, Flavobacteriales; one family, Weeksellaceae; and one genus, Chryseobacterium) was significantly different, with all showing high abundance. In the 31# groups, two phyla (Firmicutes and Actinobacteriota) and their branches (two classes, three orders, three families, and three genera) as well as one family (Beijerinckiaceae) and its branch (one genus) exhibited significant differences, with Bacilli and Firmicutes showing higher abundance. The 38# groups showed significant differences in two orders (Pseudomonadales and Streptomycetales) and their branches (two families, two genera, and one species), with high abundances in Pseudomonadales, Pseudomonadaceae, and Pseudomonas. In 64# groups, one phylum (Proteobacteria) and its branches (two classes, three orders, two families, and two genera) were significantly different, with high abundances of Proteobacteria and Gammaproteobacteria.
For endophytic fungi, Figure 10b and Figure 11b demonstrate that the biomarkers in the CK groups were significantly higher than those in the infected groups, which was the opposite of the trend observed for endophytic bacteria. In the CK groups, classifications with significant differences included one kingdom and its branch (one phylum, one class, one order, one family, and one genus), one phylum (Basidiomycota), two classes (Saccharomycetes and Eurotiomycetes), and the branches of Saccharomycetes (one order, one family, three genera, and one species). Additionally, two orders (Helotiales and Trichosphaeriales) and their branches (two families and two genera) were identified, with Saccharomycetes and Saccharomycetales demonstrating higher abundances than the others. In the 31# groups, one class (Sordariomycetes) with its branch (one order, one family, and one genus) was significantly different, all showing high abundance. Similarly, in the 64# groups, one class (Dothideomycetes) with its branch (one order, one family, and one genus) was significantly different, with high abundance.

4. Discussion

4.1. Endophyte Diversity in Healthy and Infected Avocados

Few studies have investigated the impact of anthracnose pathogens on endophyte community structure in avocados. However, research on other crops, such as wheat (crown rot) [38], citrus (Huanglong disease) [39], and potato (scab) [40], has demonstrated significant alterations in the endophytic colony structure due to pathogenic bacterial and fungal infection. High-throughput sequencing was used to analyze the structure and diversity of endophytic microbial communities in the healthy and anthracnose-infected postharvest avocado fruits. It was revealed through analyses of the alpha and beta diversity that pathogenic fungal infection significantly affected the avocado endophytes. These results indicated that the diversity and richness of endophytic bacteria in healthy avocado fruits (CK) were lower than those in the infected groups, while the endophytic fungi presented no such difference. These results are consistent with those of Peng et al. [27] on mulberry fruits. Li et al. [41] observed the decreased internal beneficial fungi and reduced diversity in infected plants, with increasing levels of pathogenic fungi. The anthracnose pathogens damaged the normal tissues of avocado fruits, reducing the fruit defenses and allowing more external bacteria to infiltrate. Consequently, the diversity of endophytic bacteria in the infected avocado fruits increased [42].

4.2. Structural Composition and Differences of Endophytic Bacterial Communities in Avocados

The endophytic bacterial community structure of healthy avocados was simpler than that of infected avocados, a pattern that contrasted with the diversity observed in endophytic fungi. Proteobacteria and Bacteroidota predominated in healthy avocados, whereas Firmicutes and Actinobacteriota were introduced in infected avocados. The Bacteroidota phylum exhibited a consistent decline and demonstrated the greatest variability in infected avocados, indicating a higher sensitivity to the anthracnose pathogens than other phyla. This result aligns with the study by Wang et al. [43], who identified these phyla as predominant in avocado fruits, and these phyla are similar to the main bacterial phyla in the human gut [44]. Proteobacteria, one of the largest divisions within prokaryotes, includes 6 classes and over 200 genera [45,46,47]. Firmicutes was noted by Ludwing et al. [48] to comprise more than 240 genera. Additionally, Li et al. [49] reported that chitin application significantly increased the relative abundances of Firmicutes and Actinobacteriota and reduced symptoms caused by root-knot nematodes. At the genus level, significant differences were observed in avocado fruits before and after infection. The healthy group contained only 3 genera, while the infected groups had between 8 and 10 genera. These genera are commonly found in soil, plants, food, and the environment. Some species within Chryseobacterium [50], Paenibacillus [51], Curtobacterium [52], Streptomyces [53], Pseudomonas [54], and Methylobacterium-Methylorubrum [55] were found to be known spoilage bacteria and plant pathogens, whereas the species in Stenotrophomonas [56], Bacillus [57], Paenibacillus [51], and Ochrobactrum [58] were found able to cause diseases in humans. Among the three infected groups, strain 64# exhibited a distinct colony structure compared to strains 31# and 38#, with higher relative abundances of Stenotrophomonas, Sphingobacterium, and Ochrobactrum and lower relative abundances of Chryseobacterium, Curtobacterium, and Pseudomonas. This variation may be due to differences in pathogen strains and infection severity. Further research is likely be required.

4.3. Structural Composition and Differences of Endophytic Fungal Communities in Avocados

The composition of endophytic fungal phyla in avocados was simpler than that of endophytic bacteria. Previous studies have identified Ascomycota and Basidiomycota as the main endophytic fungi in avocados [10] and noted their dominance among fungi in other fruits [59,60]. Specifically, the phylum Ascomycota comprises 805 species across 352 genera, whereas Basidiomycota includes 21 species in 17 genera [61]. Additionally, the Anthophyta phylum was observed in this study, which is consistent with the findings of Wang et al. [43], who reported its presence in blueberry fruit. Notably, the abundance of Anthophyta decreased significantly in both infected avocados and post-storage blueberries. Following the anthracnose pathogen influence, the relative abundance of Anthophyta and Basidiomycota fell below 0.1%. These results suggest that these endophytic phyla could serve as the sensitive indicators for anthracnose. At the genus level, the endophytic fungi were highly abundant in the healthy avocados, with 10 genera exhibiting relative abundances greater than 0.3%. Some of these genera, including Candida, Oidiodendron, Nigrospora, Talaromyces, Colletotrichum, Pestalotiopsis, Lasiodiplodia, and Fusarium [43,62,63,64,65,66], are known spoilage and pathogenic fungi. These genera are common pathogens in various fruits, causing diseases such as stem blight, gray mold, canker lesions, shoot dieback, and anthracnose [67,68,69,70]. An increasing number of records have identified these renowned pathogens within asymptomatic hosts, although explanations for this phenomenon are not immediately available [71,72,73]. The healthy avocados contained a variety of pathogenic fungi without exhibiting disease, likely due to the plant–microbe synergism that helped the plant resist the pathogenic bacteria, supported by a rich endophytic fungal community antagonistic to Anthracnose spp. [74,75,76]. The infection with anthracnose pathogens led to a rapid increase in the relative abundance of the Colletotrichum genus (78.68–86.28%), followed by Lasiodiplodia (11.17–20.12%), with an observed inverse proportionality between the two genera. The endophytic fungal community structure in the infected avocados was drastically reduced, consistent with the findings of Blaustein et al. [77], which suggested that the variations in endophytic flora could directly correlate with the differences in disease severity. The high levels of pathogen infection may destabilize the original endophytic fungal community and increase the relative abundance of Anthracnose genera, leading to dominant anthracnose disease in avocados.
Recently, various actinomycetes, bacteria, and fungi, including Streptomyces spp., Bacillus megaterium, Bacillus subtilis [78], Pseudomonas fluorescence, Pseudomonas putida [79], Penicillium spp. [80], Phoma spp., and Trichoderma spp. [81], have proven effective as biocontrol agents. These fungi that are found in healthy avocados, such as Eucalyptus, Candida, and Kluyveromyces, could be combined into a composite fungal agent with the potential to inhibit anthracnose through synergistic effects. Future research should focus on developing and assessing the efficacy of bacterial and fungal microbial agents for anthracnose inhibition.

5. Conclusions

This study preliminarily explored the structure and diversity of endophytic microbial in both healthy and anthracnose-infected postharvest avocado fruits. The results revealed that there were differences in the endophytic microbial communities of avocado fruits before and after infection. The endophytic bacterial community was primarily composed of 4 phyla and 10 genera, and the endophytic fungal community was characterized by 3 phyla and 10 genera. The key biomarkers included two phyla, two classes, two orders, two families, and two genera in endophytic bacteria. The important biomarkers were one class, three orders, two families, and two genera in endophytic fungi. Further experimental research is required to elucidate the reasons for these differences. These findings provide the foundational data for constructing microbial systems related to postharvest avocado fruits anthracnose and developing composite microbial agents. These studies would have potential positive implications for the biological control of postharvest avocado fruits anthracnose.

Author Contributions

Writing—original draft preparation, X.C.; software, H.S. and Z.W.; writing—review and editing, S.Z. and W.W.; funding acquisition, J.C. and X.T. (Xinghao Tu); methodology, P.H., Z.J., T.Z., T.D. and J.Q.; resources, X.T. (Xiuhua Tang). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Foundation of Research Project from Key Laboratory of Tropical Fruit Biology, Ministry of Agriculture & Rural Affairs (grant number 2023KFKT-01); Foundation of Fundamental Research Project from Guangxi Academy of Agricultural Sciences (grant number 2021YT159 and 2023YM20); and Foundation of Research Project from Science and Technology Vanguard (grant number 202404-08); Central Public-interest Scientific Institution Basal Research Fund (grant number 1630062022001).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors are grateful for the support of the Guangxi Key Laboratory of Fruits and Vegetables Storage-Processing Technology, Guangxi Academy of Agri-cultural Sciences.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Healthy Avocado Groups (CK).
Figure 1. Healthy Avocado Groups (CK).
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Figure 2. Infected Avocado Groups. (a) Colletotrichum sp. strain No. 31 (31#). (b) Colletotrichum sp. strain No. 38 (38#). (c) Colletotrichum sp. strain No. 64 (64#).
Figure 2. Infected Avocado Groups. (a) Colletotrichum sp. strain No. 31 (31#). (b) Colletotrichum sp. strain No. 38 (38#). (c) Colletotrichum sp. strain No. 64 (64#).
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Figure 3. Distribution of endophytic sequences. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Figure 3. Distribution of endophytic sequences. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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Figure 4. Annotation result statistics of ASVs. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent infected groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Figure 4. Annotation result statistics of ASVs. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent infected groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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Figure 5. Rarefaction curve of endophytes. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Figure 5. Rarefaction curve of endophytes. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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Figure 6. Species accumulation boxplot of endophytes. (a) Endophytic Bacteria. (b) Endophytic Fungi.
Figure 6. Species accumulation boxplot of endophytes. (a) Endophytic Bacteria. (b) Endophytic Fungi.
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Figure 7. NMDS of endophytes. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Figure 7. NMDS of endophytes. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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Figure 8. Histogram of relative abundance of species in the endophytic bacteria. (a) Phylum Level. (b) Genus Level. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Figure 8. Histogram of relative abundance of species in the endophytic bacteria. (a) Phylum Level. (b) Genus Level. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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Figure 9. Histogram of relative abundance of species in the endophytic fungi. (a) Phylum Level. (b) Genus Level. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Figure 9. Histogram of relative abundance of species in the endophytic fungi. (a) Phylum Level. (b) Genus Level. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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Figure 10. LEfSe Cladogram of Endophytes. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Figure 10. LEfSe Cladogram of Endophytes. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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Figure 11. Histogram of LDA score distribution. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Figure 11. Histogram of LDA score distribution. (a) Endophytic Bacteria. (b) Endophytic Fungi. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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Table 1. Statistical results of endophytic bacterial tags.
Table 1. Statistical results of endophytic bacterial tags.
SampleReadsEffective TagsMerged
(%)		GCQ20Q30MeanError
CK-1146,146133,00291.0152.7098.9596.070.43
CK-2140,549128,32591.3053.1598.9196.000.44
CK-351,01846,70791.5553.2798.9696.160.43
S31-1148,218135,60991.4954.5398.9296.020.44
S31-2132,163121,49091.9254.5798.9796.160.43
S31-3107,98899,41792.0654.2798.9596.110.43
S38-1148,125135,52891.5054.0498.9396.070.44
S38-266,23960,93792.0054.0998.9396.090.44
S38-3147,885133,27490.1254.1198.9396.060.44
S64-1148,010134,51590.8854.3398.8795.850.46
S64-2147,765132,01889.3454.3398.8395.730.48
S64-3147,694133,08390.1154.3298.8895.910.46
Note: CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Table 2. Statistical results of endophytic fungal tags.
Table 2. Statistical results of endophytic fungal tags.
SampleReadsEffective TagsMerged
(%)		GCQ20Q30MeanError
CK-1140,771128,28491.1353.5798.9896.320.37
CK-2136,655127,33693.1854.1699.2097.050.29
CK-3137,931127,80092.6653.5199.1496.840.31
S31-169,49365,27193.9252.6599.2797.330.26
S31-2139,577129,98793.1352.6099.2497.220.27
S31-3100,34594,60394.2852.5999.3197.430.25
S38-1137,993128,96693.4652.6299.2697.310.26
S38-2147,063137,56393.5452.6199.2597.270.27
S38-3137,068127,69893.1652.6099.2997.380.26
S64-1142,588133,04493.3152.2999.2797.310.26
S64-2146,918137,94193.8952.3099.2597.280.27
S64-3122,538115,42194.1952.2799.2997.380.26
Note: CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Table 3. Diversity indexes of endophytic bacterial communities in avocado fruits.
Table 3. Diversity indexes of endophytic bacterial communities in avocado fruits.
SampleChao1ACEShannonSimpsonCoverage
CK-1653.92654.212.440.8399.91
CK-2580.56570.491.900.6899.92
CK-3433.21434.872.220.7799.80
S31-1900.60878.103.270.9099.93
S31-2911.04907.293.570.9399.92
S31-3863.67852.602.970.8299.88
S38-1844.82845.832.850.8499.94
S38-2759.64758.122.870.8499.79
S38-3862.42831.992.970.8599.93
S64-1848.88839.712.100.5899.91
S64-2861.99857.842.190.6099.89
S64-3833.72823.871.910.5299.90
Note: CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Table 4. Diversity indexes of endophytic fungal communities in avocado fruits.
Table 4. Diversity indexes of endophytic fungal communities in avocado fruits.
SampleChao1ACEShannonSimpsonCoverage
CK-1313.23320.123.160.9099.96
CK-2367.44369.683.680.9499.97
CK-3400.67370.383.560.9399.96
S31-1274.93277.040.860.3399.90
S31-2250.41254.620.650.2499.96
S31-3276.69288.810.720.2799.93
S38-1174.33191.630.700.3399.96
S38-2290.02295.100.730.3299.94
S38-3202.36215.820.690.3199.96
S64-1265.10265.690.950.4199.95
S64-2281.22292.500.980.4299.95
S64-3258.29260.000.950.4199.95
Note: CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Table 5. Major phyla identified for endophytic bacteria (%).
Table 5. Major phyla identified for endophytic bacteria (%).
PhylumCKS31S38S64
Proteobacteria76.61 ± 4.09 a53.33 ± 9.69 a79.27 ± 2.30 a88.49 ± 1.38 a
Bacteroidota21.31 ± 3.35 b 4.16 ± 1.02 c7.74 ± 0.40 b5.66 ± 0.48 b
Firmicutes1.06 ± 0.42 c27.4 ± 2.93 b5.08 ± 1.01 c4.16 ± 0.89 b
Actinobacteriota0.69 ± 0.46 c13.89 ± 6.66 c7.53 ± 0.89 bc1.30 ± 0.27 c
Note: Means with different lowercase letters in the same column are significantly different (p < 0.05). Same as below. CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Table 6. Major genera identified for endophytic bacteria (%).
Table 6. Major genera identified for endophytic bacteria (%).
GenusCKS31S38S64
Stenotrophomonas38.16 ± 16.50 a25.02 ± 14.82 a23.82 ± 1.33 b66.44 ± 3.35 a
Chryseobacterium20.02 ± 3.66 b3.86 ± 0.97 de7.24 ± 0.43 d0.94 ± 0.17 e
Others10.65 ± 1.19 bc9.85 ± 1.18 cd11.94 ± 0.56 c10.05 ±0.64 b
Bacillus0.09 ±0.02 c11.14 ± 3.49 cd4.07 ± 0.71 e2.89 ± 0.64 ef
Paenibacillus0.06 ± 0.02 c15.56 ± 1.85 bc0.10 ± 0.04 g0.95 ± 0.18 ef
Sphingobacterium0.05 ± 0.03 c0.03 ± 0.01 e0.05 ± 0.03 g4.60 ± 0.46 de
Curtobacterium0.03 ± 0.01 c10.34 ± 4.93 cd3.37 ± 0.47 e0.36 ± 0.11 f
Ochrobactrum0.03 ± 0.01 c0.07 ± 0.02 e0.07 ± 0.01 g6.83 ± 1.24 c
Streptomyces0.03 ± 0.02 c0.42 ± 0.27 e2.12 ± 0.41 f0.50 ± 0.07 f
Pseudomonas30.86 ± 11.92 a21.48 ± 4.85 ab46.35 ± 1.12 a6.31 ± 0.50 cd
Methylobacterium-Methylorubrum0.02 ± 0.01 c2.22 ± 1.19 de0.86 ± 0.24 g0.11 ± 0.01 f
CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Table 7. Major phyla identified for endophytic fungi (%).
Table 7. Major phyla identified for endophytic fungi (%).
PhylumCKS31S38S64
Ascomycota70.18 ± 2.25 a99.87 ± 0.03 a99.92 ± 0.03 a99.86 ± 0.05 a
Anthophyta23.71 ± 2.65 b0.06 ± 0.02 b0.04 ± 0.01 b0.09 ± 0.09 b
Basidiomycota5.25 ± 0.48 c0.06 ± 0.01 b0.04 ± 0.02 b0.05 ± 0.01 b
Note: CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
Table 8. Major genera identified for endophytic fungi (%).
Table 8. Major genera identified for endophytic fungi (%).
GenusCKS31S38S64
Others29.91 ± 8.23 a1.24 ± 0.20 c0.89 ± 0.07 c0.80 ± 0.05 c
Eucalyptus22.49 ± 3.36 ab0.06 ± 0.02 c0.04 ± 0.01 d0.08 ± 0.04 d
Candida14.46 ± 6.49 bc0.11 ± 0.03 c0.04 ± 0.03 d0.08 ± 0.04 d
Kluyveromyces11.63 ± 8.50 cd0.07 ± 0.03 c0.03 ± 0.02 d0.06 ± 0.02 d
Talaromyces6.28 ± 5.26 cd0.04 ± 0.00 c0.02 ± 0.01 d0.04 ± 0.02 d
Oidiodendron5.89 ± 8.49 cd0.03 ± 0.01 c0.01 ± 0.01 d0.03 ± 0.01 d
Colletotrichum3.88 ± 1.05 de86.28 ± 2.57 a83.24 ± 0.98 a78.68 ± 0.90 a
Nigrospora3.21 ± 1.33 de0.04 ± 0.01 c0.01 ± 0.01 d0.03 ± 0.00 d
Pestalotiopsis1.34 ± 0.79 e0.03 ± 0.01 c0.01 ± 0.01 d0.03 ± 0.01 d
Lasiodiplodia0.58 ± 0.51 e11.17 ± 2.16 b15.67 ± 1.07 b20.12 ± 0.78 b
Fusarium0.34 ± 0.18 e0.92 ± 0.13 c0.04 ± 0.02 d0.05 ± 0.02 d
Note: CK represents the healthy group. S31, S38, and S64 represent the groups infected by anthracnose strains 31#, 38#, and 64# in sequence.
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MDPI and ACS Style

Chen, X.; Jiang, Z.; He, P.; Tang, X.; Song, H.; Zhang, T.; Wei, Z.; Dong, T.; Zheng, S.; Tu, X.; et al. Effects of Anthracnose on the Structure and Diversity of Endophytic Microbial Communities in Postharvest Avocado Fruits. Agronomy 2024, 14, 2487. https://doi.org/10.3390/agronomy14112487

AMA Style

Chen X, Jiang Z, He P, Tang X, Song H, Zhang T, Wei Z, Dong T, Zheng S, Tu X, et al. Effects of Anthracnose on the Structure and Diversity of Endophytic Microbial Communities in Postharvest Avocado Fruits. Agronomy. 2024; 14(11):2487. https://doi.org/10.3390/agronomy14112487

Chicago/Turabian Style

Chen, Xi, Zhuoen Jiang, Peng He, Xiuhua Tang, Haiyun Song, Tao Zhang, Zhejun Wei, Tao Dong, Shufang Zheng, Xinghao Tu, and et al. 2024. "Effects of Anthracnose on the Structure and Diversity of Endophytic Microbial Communities in Postharvest Avocado Fruits" Agronomy 14, no. 11: 2487. https://doi.org/10.3390/agronomy14112487

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